HRP20010113A2 - Process for purifying human papillomavirus virus-like particles - Google Patents

Abstract

A process for purifying papillomavirus virus-like particles (VLPs) includes the step of passing a partially purified VLP-containing solution through a hydroxyapatite chromatography column. The VLPs are then eluted using a buffer containing phosphate anion. The advantages of this method include the recovery of a high yield of intact VLPs.

Description

Field of invention

The subject invention relates to the process of preparation and purification of papilloma virus (HPV) virus-like particles (VLP), which can be used as a component in a vaccine.

Background of the invention

Papillomavirus infections occur in a variety of animals, including humans, sheep, dogs, cats, rabbits, monkeys, snakes, and cows. Papillomaviruses infect epithelial cells, generally causing benign epithelial or fibroepithelial tumors at the site of infection. Papilloma viruses are species-specific infectious agents; human papillomavirus cannot infect other animals.

Papilloma viruses can be classified into different groups, according to the host they infect. Human papillomaviruses (HPV) are further divided into more than 70 species, based on DNA sequence homology (for review see Human Papillomavirus and Cancer, H. Pfister (ed.), CRC Press, Inc., 1990). The types of papillomaviruses are apparently immunogenic by type, insofar as neutralizing immunity to infection with one type of papillomavirus does not guarantee immunity against another type of papillomavirus.

Papillomaviruses are small (50-60 nm), non-enveloped, icosahedral, DNA viruses that encode up to eight early and two late genes. The open reading frames (ORFs) of the virus genome are designated E1 to E7 and L1 and L2, where "E" (early) indicates early and "L" (late) late. L1 and L2 encode viral capsid proteins. Early (E) genes are related to functions such as viral replication and cell transformation.

The L1 protein is the main capsid protein and has a molecular weight of 55-60kDa. L2 is a minor capsid protein, having a predicted molecular weight of 55-60 kDa, and an apparent molecular weight of 75-100 kDa as determined by polyacrylamide gel electrophoresis. Immunological data show that most of the L2 protein is internal to the L1 protein. L2 proteins are well conserved in various papillomaviruses, especially the 10 basic amino acids at the C-terminus. The L1 ORF is well conserved among various papillomaviruses.

Recombinant L1 protein is made in various hosts, and under the right conditions assembles into a virus-like particle (VLP), alone or in combination with L2. VLPs are candidates for commercial vaccines. However, to be useful in a human vaccine, VLPs must be highly purified and free from host cell contamination. In the past, contaminating biomolecules were removed by flow ultrafiltration in diafiltration mode. However, this method resulted in proteolytic degradation of HPV L1. It would be desirable to have such an LI protein purification procedure, which gives a non-degraded product of high purity.

Summary of the invention

This invention relates to a process for the purification of recombinant papillomavirus (HPV) virus-like particles (VLP), which consists of the following steps: contacting a partially purified VLP containing cell lysate with a hydroxyapatite medium in a chromatography column, under such conditions that VLPi bind to hydroxyapatite medium; and eluting the bound VLPa with a solution consisting of phosphate anions; and separation of eluted VLPs.

The purification procedure can be used with VLPs consisting mainly of L1 protein, and can be used with VLPs containing both L1 and L2 proteins.

Additionally, it can be used with chimeric VLPs, i.e. those containing L1 protein and L2 protein fusion. In general, for vaccine use, VLPs containing only L1 are more suitable.

The procedure is applicable to VLPe from practically any type of papillomavirus. Human papilloma virus (HPV) is preferably used. Preferred types of HPV are those known to cause the most severe diseases and conditions, including HPV type 6a, HPV type 6b, HPV type 11, HPV type 16, HPV type 18, HPV type 31, HPV type 33, and HPV type 45.

Generally, a host cell is transformed with a vector encoding L1 or L1 and L2 proteins, or an L1 and L2:fusion protein.

As used in the specification and claims, the term "L2:fusion protein" means that the DNA encoding the L2 protein is operably linked to another DNA encoding the desired protein, preferably another HPV protein, such as E1, E2, E3, E4, E5, E6 or E7. The L2 portion of the fusion protein can be full-length, or it can have deletions and/or truncations. Examples can be found in US provisional patent application S.N. 60/096, 638, (lawyer application number 20276PV, which is hereby incorporated in its entirety) which was filed here.

The host cell can be any host cell that is readily isolated in culture, as is known in the art, including yeast (Saccharomyces cerevisiae), insect, bacterial or mammalian cells. Yeast cells are particularly preferred.

The vector may also contain other elements known in the art, such as transcriptional and translational control elements and/or marker genes. Expressed L1, L1 and L2, or L1 and L2:fusion proteins spontaneously assemble into VLPs. Host cells are lysed in a conventional manner and then the cell lysate is partially purified.

The partial purification step may include known purification steps, and is not critical in this invention. For example, the cell lysate may be subjected to a microfiltration process and at least one chromatography step, such as cation exchange chromatography.

It has been found in accordance with the present invention that a chromatography step using hydroxyapatite as the column medium, followed by elution with a phosphate anion buffer solution, removes large amounts of contaminants from partially purified cell lysates.

Specifically, most contaminating biomolecules, including DNA, lipids, and proteins, were found to be removed from the lysate.

In accordance with the present invention, the final purified VLP preparation is generally at least 75% pure, preferably at least 80% pure, and most preferably at least 90% pure, as measured using an SDS/PAGE assay.

Almost any commercially available hydroxyapatite column material can be used in this invention. It is preferred to use ceramic hydroxyapatite with a particle size of approximately 20-50 μm and a pore size of approximately 800 A. One such commercially available hydroxyapatite is sold by BioRad as "Ceramic Hydroxyapatite, Type II". However, others are equally effective.

In preparation for the chromatography step in the purification process, it is recommended that the column be loaded with a buffer of pH 6-8, preferably around 7. The preferred buffer is 50 mM MOPS [3-(N-morpholine)propanesulfonic acid] with pH 7.0 and also with 1.25 MNaCl.

Other buffer systems that may also be used are known in the art and include:

MES [2-(N-morpholino)ethanesulfonic acid];

BIS-TRIS [bis-(2-hydroxyethyl)amino]tris-(hydroxymethyl)methane];

ADA [N-2-acetamidoiminoacetic acid, monosodium salt];

ACES [N-2-acetamido-2-aminoethanesulfonic acid, monosodium salt];

PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)];

MOPSO [(3-N-morpholino)-2-hydroxypropane-sulfonic acid];

BIS-TRIS PROPHAN [1,3-bis[tris(hydroxymethyl)methyl-amino]propane];

BES [N,N-bis-(2-hydroxymethyl)-2-amino-ethanesulfonic acid];

TES [N-tris-(hydroxymethyl)methyl-2-aminoethane sulfonic acid]; and

2-2([2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethane sulfonic acid];

HEPES [N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid];

DIPSO [3-(N,N-bis-(2-hydroxymethyl)-amino)-2-hydroxy propanesulfonic acid];

TAPSO [3-N-tris(hydroxymethyl)methylamino]-2-hydroxy-propanesulfonic];

TRIS [tris-(hydroxymethyl)-aminoethane];

HEPPSO [N-(2-hydroxyethyl)-piperazine-N'-[2-hydroxy-propanesulfonic acid];

POPSO [(piperazine-N,N'-bis-[2-hydroxypropanesulfonic acid]);

EPPS [N-[2-hydroxyethyl)-piperazine-N'-[3-propanesulfonic acid and HEPPS];

TEA [triethanolamine];

TRICINE [N[tris-(hydroxymethyl)methyl]glycine];

BICINE [N,N-bis-(2-hydroxyethyl)-glycine];

TAPS [3-{[tris-(hydroxymethyl)methyl]amino}propane sulfonic acid];

imidazole;

HEPPS [N-2-Hydroxyethylpiperazine-N'-3-propanesulfonic acid];

glycine amide, hydrochloride;

glycylglycine;

citrate;

acetate;

and succinate buffers.

Partially purified VLP in buffered solution was brought into contact with hydroxyapatite medium under conditions that allow VLPu binding to hydroxyapatite. These conditions include a wide range of temperatures; room temperature is preferred. The flow rate can vary considerably, and the preferred range is 90 cm/hour.

After the binding of VLPa to hydroxyapatite, the next step is to separate the purified VLPa from hydroxyapatite with an elution buffer. A preferred elution buffer solution contains a phosphate anion, such as a sodium or potassium phosphate solution. Preferred molar ranges are from about 0.05 M to about 1 M, and most preferably about 0.2 M. The pH of the elution buffer should be from about pH 6-8, and most preferably pH 7.

Other advantages of the methods of the present invention include:

(a) no requirement for special chromatography equipment and techniques;

(b) the method is fast, does not require buffer changes, and

(c) the method gives an excellent yield of HPV L1.

The following non-limiting Examples are presented to better illustrate the present invention.

EXAMPLES

EXAMPLE 1

PREPARATION OF PARTIALLY PURIFIED LYSATE

Yeast cells transformed to express VLP were harvested and frozen at -70°C. The frozen yeast cell solution was allowed to thaw for about 3 hours at room temperature and then for approximately 18 hours at 4°C. BENZOSANE® (Nycomed Pharma A/S, Copenhagen, Denmark) (2.8 x 105 units/mL and 0.21 mg protein/mL) was added to the cell solution to a final concentration of 750 units per gram wet cell weight, and in one experiment was reduced to 335 units per gram of wet cell weight. Cells were mixed for 15 minutes, then disrupted using two passes through a sanitized APV Gaulin 30CD homogenizer at a chamber pressure of 14,500 to 16,000 psi, resulting in 95% cell disruption. The remaining lysate was gently stirred for 18 hours at 4°C.

Purification by microfiltration. The cell lysate was purified by cross-flow microfiltration in diafiltration mode, as described. The lysate was transferred to a sterile processing vessel with a radius and inch slit, and an exit port. The microfilter is a 0.65 micron pore, 5 square foot hollow fiber filter cartridge (A/G Technologies #CFP-6-D-8A, Needham, MA), housed in an A/G Technologies FlexStand® Benchtop Pilot hollow fiber system . The residue was diafiltered with 3 volumes of diafiltration buffer (below) to obtain a purified lysate. Diafiltration buffer is 0.2 M (Na+) MOPS, pH 7.0 + 0.4 M NaCl.

Chromatography of purified lysate. The purified lysate was fractionated by column chromatography using POROS® 50 HS strong cation exchange chromatography resin (PerSeptive Biosystems, Framingham, MA) packed in a chromatography column. The column was washed with 0.5 N NaOH before use. The column was equilibrated with HPV difiltration buffer [0.2M (Na+) MOPS, pH 7.0 + 0.4 M NaCl] at room temperature. Cold (4°C) clarified lysate was pumped onto the column at a rate of 125 mL/minute and the column was washed with 8 column volumes of HPV column buffer A at room temperature [0.05M (Na+) MOPS, pH 7.0 + 0.5 M NaCl] at 125 mL/minute with a linear gradient from 100% HPV column buffer A to 100% column buffer B [0.05M (Na+) MOPS, pH 7.0 + 1.5 M NaCl]. The total linear gradient was 10 column volumes and collected in 10 equal volume fractions. Following the gradient, the column was washed with two column volumes at room temperature of HPV column buffer B at 125 mL/minute, which were collected in two additional fractions. Fractions were collected in sterile 2-liter plastic bottles and left at 4°C. Fractions with the last UV-absorbing peak (A280 nm and A230 nm) in the gradient were collected, filtered using a MILLIPAK-200 consumable filter unit (Millipore, Bedford, MA) and left at 4°C.

EXAMPLE 2

HYDROXYPATITE CHROMATOGRAPHY

All steps were performed at room temperature. A chromatography column (13 mm ID x 36), packed with ceramic hydroxyapatite, type II (BioRad cat.#7320081, Hercules, CA), was previously equilibrated in 50 mM MOPS, pH 7.0 + 1.25 M NaCl. The partially purified solution of HPVa from Example 1 was placed on the column at a linear flow rate of 90 cm/hour. After sample application was complete, the column was washed with eight column volumes of pre-equilibration buffer until the optical density of the column effluent reached near zero. The HPV vaccine product was eluted with a linear gradient from 0% to 100% elution buffer (0.2 M sodium phosphate, pH 7.0 + 1.25 M NaCl), also with a linear flow rate of 90 cm/hour. The total gradient volume was four column volumes. Fractions containing the vaccine product were determined by RIA and the Bradford protein assay. The protein concentration in the product was 100 mg/mL.

Assays: Bradford protein assays were performed using Coomassie Plus assay reagent (Pierce, Rockford, IL) using bovine serum albumin (BSA) as a standard. Lowry protein assays were performed according to the procedure of Lowry et al. 195 U. Biol. Chem. 193:265-270, using BSA as a calibration standard. The antigen was tested by a multi-layer ELISA test using a monoclonal antibody that recognized the conformational epitope of VLPa. Microfilter plates are covered with polyclonal goat anti-HPV VLP antibodies. Standard and test samples were diluted with PBS, with 1% w/v BSA, 0.1% TWEEN-20, and 0.1% sodium azide, and added to the wells where the antigen was captured by antibodies bound to the plates. Monoclonal anti-HPV LI VLP antibody (Chemicon, Temecula, CA) was added to the wells to bind antigen captured by antibodies bound to the plates. Monoclonal anti-HPV LI VLP antibodies were detected using horseradish peroxidase conjugates with anti-mouse IgG antibodies. A chromogenic substrate for horseradish peroxidase, 3,3',5,5'-tetramethylbenzedine (Pierce) was then added and the absorbance at 450 nm, which is proportional to the concentration of LI VLPa in the sample, was measured.

The dynamic capacity of the column for the vaccine product was 2.9 mg per mL of resin by Bradford, and 4.6 mg per mL of resin by RIA. Recovery through this step was 90% by Bradford protein assay or 82% by RIA, when the column was loaded to 100% capacity. The recovery dropped to 63% by Bradford and 50% by RIA, when the column was loaded to 8% capacity.

EXAMPLE 3

Removal of other biomolecules The HPV L1 sample prepared as described in Examples 1 and 2 was tested for the presence of DNA, using a PCR assay. The results, shown in the table below, show that this chromatography method is highly effective in removing contaminating DNA from the final product.

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EXAMPLE 4

Purification of chimeric VLP

Purification of HPV type 16 L1/L2mini/E2 chimeric VLP

Construction of the modified L2 gene

YP3 Vector (minimum L2)

This vector harbors the coding sequences for the amino-terminal 69 amino acids and the carboxy-terminal 84 amino acids (aa) of HPV 16 L2, which is fused in frame using a synthetic poly-linker that introduces unique Not I, Sac I, and Xho I restriction enzyme sites. and results in the insertion of one glutamic acid residue and the mutation of a serine residue to glutamic acid.

PCR "primers" or primers (Midland Certified Reagents) were designed to amplify L2 sequences from the native L2 gene contained in the pGallO HPV 16 LI + L2 vector.

"Examples" I (5' - CTT CCC CCC GGG C AC AAA ACA AAA TGC -3'; SEQ. ID. NO. 1) and C (5' - CTC GAG CTC GCG GCC GCC TGT ACC CGA CCC - 3'; SEQ ID NO: 2) amplified to a 265 bp sequence encoding the amino terminus of 69 aa and 23 bp of upstream untranslated sequence, which includes the Srna I restriction enzyme site.

"Primers" C modify and extend the L2 amino-terminal coding region and add Not I, Sac I, and Xho I restriction enzyme sites downstream of the L2 coding sequences.

"Primers" A (5' - GCG GCC GCG AGC TCG AGG GTT ATA TTC CTG CAA ATA CAA-3'; SEQ. ID. NO. 3), C and D (5' - CCC TCC AGA TCT CTA GGC AGC CAA AGA GAC ATC TG-3'; SEQ ID NO: 4) amplified to a 285 bp sequence encoding the carboxy terminus of 84 aa L2 plus 6 bp that added a Bgl II restriction enzyme site. "Primer" A also added a 17 bp sequence with Not I, Sac I and Xho I sites upstream of the L2-coding sequence.

The minimal construct for L2 expression was assembled through complementary sequences, added using "primers" A and C. Isolated DNA products I/C and A/D amplified by the above reactions were both used in a PCR reaction that included I and D oligos as amplification " examples". To enable joining of fragments through their 17 bp complementary sequences, three PCR cycles were performed at an annealing temperature of 37°C, followed by 15 cycles at 57°C. The resulting amplification product was blunt-ended ligated into pcrScript (Stratagene, LaJolla) and transformed into XL-1 blue MRF cells (Stratagene, LaJolla). Positive clones were identified by PCR using "primers" I and D, and confirmed by restriction digest analysis. The construct was then verified by automated sequence analysis (Perkin Elmer, Inc., Foster City, CA).

Plasmid DNA from the corresponding isolate was then digested with Sma I and Bgl II; a fragment of approximately 0.5 kilobase pairs (kb) was gel purified and ligated with a 15 kb Srna I and Bgl II pGALl 10 HPV16 LI vector fragment. Competent DH5 E. coli cells (Gibco BRL, Rockville, MD) were transformed into ligation mixture and transformants selected on LB ampicillin plates (Remel, Lenexa, KS). Clones were initially screened by PCR using primers D and I to amplify portions of L2; suitable clones were then confirmed by restriction digestion analysis. Candidate clone YP3#1 was verified by sequence analysis, as noted above.

YP3#1 was then used as a basis for the construct, in which genes encoding HPV16 E1, E2 or E7 open reading frames were inserted.

Insertion of HPV E protein-coding genes

The gene encoding HPV 16 E2 was obtained by PCR amplification of an HPV 16 positive clinical sample, which was then directly inserted into the subclonal vector pCRII (Stratagene, La Jolla, CA) and sequence verified as described above. The E2 gene was then modified as follows:

1) Framework Xho I, Nae I and Not I containing DNA sequences were added to the amino terminus of E2. Additionally, sequences containing Not I, Nae I, and Xho I were added to the carboxyl terminus of E2 to facilitate insertion into E2, at the Not I and Xho I sites.

2) The DNA sequences were changed by PCR mutagenesis to encode alanine residues, coding for glutamic acid residues 39 and isoleucine 73. This was done to inactivate E2 protein function. The modified HPV16 E2 gene described above was digested with Not I and Xho I, and

ligated with similarly digested YP3#1 vector. Transformants, which contained properly inserted E2 sequences, were selected by PCR, and the sequence verified.

The same strategy was used for the genes encoding HPV16 E1 and HPV16 E7. For E1, glycine 482 was changed to aspartic acid; for E7, cysteine 24 and glutamic acid 26 are both changed to glycine, to inactivate protein function. The resulting constructs were then used to transform yeast for expression analysis.

EXAMPLE 5

Identification and growth of yeast expressing chimeric VLPs

Plasmid DNA YP3#1 and derivatives described above were used to transform Saccharomyces cervisiae (MATa, Ieu2-04, prbl::HIS3, mnn9::URA3, cir°) by the spheroplast method (Hinnen et al., 1978, Proc. Natl. Accad Sci USA 75:1929-1933). Transformed spheroplasts were plated on selective medium (leucine minus) (Remel, Lenexa, KS). Clones were isolated through two rounds of colony selection. Small liquid cultures of candidate clones were grown to high cell density on galactose medium. Crude extracts were prepared by vigorous mixing with glass beads, followed by centrifugation. Purified extracts were analyzed for the expression of L1 and L2 components, and VLPi by various methods including SDS PAGE, ELISA, immunoblotting, EIA, using monoclonal antibodies or monospecific polyclonal antisera that recognize L1 or L2 or the amino or carboxy terminus of L2 or L1 VLPa, or E1, or E2, or E7, or some other protein or peptide linked to the modified L2. Clones that expressed the L2 component and formed VLPs were selected for further characterization. One-liter or 16-liter cultures of selected clones were grown in galactose containing medium for the preparation of chimeric VLPs.

Cell pellets were frozen at -70°C. Frozen cells (wet weight = 148g) were thawed and redissolved in 740 mL of "breaking" buffer (200 mM MOPS, pH 7, 1 mM CaCl 2 ) to obtain an approximately 20% (w/v) mixture. Nuclease BENZONASE® (Nycomed Pharma) was added at 750 units/g wet cell weight. The cell mixture was disrupted at a pressure of approximately 19,000 psi in 5 steps in an Ml 10-Y microfluidizer (Microfluidics Corp., Newton, MA). The cell mixture was collected and kept on ice during lysis. Hematocrit test showed > 80% fracture.

The remaining cell lysate was purified by microfiltration through a 0.65 micron pore size hollow texture cartridge (A/G Technologies) using a tangential flow microfiltration apparatus in diafiltration mode. The lysate was diafiltered with three volumes of 0.25 M sodium citrate, 0.2 M MOPS, pH 7.0. The antigen passed through the membrane and was collected in the Permeate.

The diafiltered 0.65 mm permeate fraction (3.9 L) was loaded onto a 325 mL column (l 1.2 cm ID x 3.3 cm) POROS® 50 HS resin (Perspective Biosystems, Cambridge, MA), equilibrated in 200 mM MOPS, pH 7, 250 mM sodium citrate. The column was washed with 8 volumes of 50 mM MOPS, 0.5 M NaCl, 5 mM sodium phosphate, pH 7 and eluted with a 10 volume linear gradient from 0.5 to 1.5 M NaCl in the same buffer. Flow-through and washed fractions were collected in bulk, while l volume of fractions was collected during elution. Colonic fractions were analyzed by Western blot method and SDS-PAGE detection with colloidal Comassie. Fractions containing mainly p55 protein were collected.

The 50HS pool of the sample was analyzed for total proteins by the BCA assay (Pierce). Based on total protein (168 mg), a column of ceramic hydroxyapatite (HA) type II (Bio-Rad) was run to yield l mL resin/2 mg protein. The column was 2.6 cm ID x 15.7 cm. The column was equilibrated in 50 mM MPPS, pH 7, 1.25 M NaCl, 5 mM sodium phosphate. 50HS pool sample (770 mL) was filtered through 0.22 mm and applied to the HA column at a flow rate of 113 cm/hour. The flow is collected in a pile. The HA column was washed with 5 volumes of equilibration buffer and eluted with 8 volumes of a linear gradient, from 5 to 200 mM sodium phosphate, pH 7 in 1.25 M NaCl. Fractions collected during elution were analyzed by Western blot method and SDS-PAGE with colloidal Coomassie detection. Fractions showing comparable purity and L1 protein enrichment were collected. The collected fractions were filtered aseptically through a 0.22 mm membrane and left at 4°C.

The residues from this process and the product were analyzed for HPV16 L1 using a specific EIA and for protein with the BCA test. The final yield of the purified product was 27 mg of protein with a specific activity of 1.00 mg L1/mg of protein. An electron microscope confirmed the presence of intact VLP particles, with a mean diameter of 32 nm. For SDS-PAGE purity analysis, an aliquot of the final product was concentrated by TCA precipitation and analyzed by Western blot and SDS-PAGE with colloidal Coomassie detection. Quantification of L1 was done using a 2.5 mg charge and yeast contaminants were quantified using a 20.0 mg charge. It was shown by densiometry that the L1 protein is > 94% homogeneous. Co-purification of L1 and L2 minje was demonstrated by specific immunoblot analysis of procedure fractions.

Claims (9)

1. A process for the purification of papillomavirus-like particles (VLP), characterized by the fact that it consists of: (a) contacting the partially purified VLP-containing cell lysate with a hydroxyapatite medium in a chromatography column, under conditions such that the VLPs bind to the hydroxyapatite medium. (b) eluting the bound VLP with a solution of phosphate anions; and (c) extraction of eluted VLPs. 2. The method according to Claim 1, characterized in that the VLPi consist mainly of L1 protein. 3. The method according to Claim 1, characterized in that the VLPi are human papillomavirus (HPV) VLPi. 4. The method according to Claim 3, characterized in that the VLPs are selected from the group consisting of: HPV type 6a, HPV type 6b, HPV type 11, HPV type 16, HPV type 18, HPV type 31, HPV type 33 and HPV type 45 . 5. The method according to Claim 4, characterized in that the eluted VLPs are at least 75% pure. 6. The method according to Claim 4, characterized in that the eluted VLPs are at least 90% pure. 7. The method according to Claim 1, characterized in that the VLPi consist of L1 protein and L2:fusion protein. 8. The method according to Claim 7, characterized in that the L2:fusion protein has an L2 portion that is less than full length. 9. Process for the preparation of a purified human papillomavirus VLP product, suitable for use in a human vaccine, which consists of: a) partial purification of the cell lysate, where the cell lysate is from yeast cells, which have been transformed to express HPV L1 VLP; (d) contacting the partially purified VLP-containing cell lysate with a hydroxyapatite medium in a chromatography column, under conditions such that the VLPs bind to the hydroxyapatite medium; c) elution of bound VLPs with a solution of phosphate anions; and d) extraction of eluted VLPs.